Diabetes Publish Ahead of Print, published online March 18, 2010

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1 Diabetes Publish Ahead of Print, published online March 18, 2010 IL-10 + Tregs regulate by linked suppression Naturally arising human CD4 T cells that recognize islet autoantigens and secrete IL-10 regulate pro-inflammatory T cell responses via linked suppression Timothy I.M. Tree 1,3, Jennifer Lawson 1, Hannah Edwards 1, Ania Skowera 1,3, Sefina Arif 1, Bart O. Roep 2 and Mark Peakman 1,3 1 Department of Immunobiology, King s College London, Guy s Hospital, London SE1 9RT, UK. 2 Department of Immunohaematology & Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. 3 NIHR Biomedical Research Centre at Guy s & St Thomas NHS Foundation Trust and King s College London Correspondence should be addressed to: Dr Timothy Tree timothy.tree@kcl.ac.uk Submitted 6 April 2009 and accepted 1 March Additional information for this article can be found in an online appendix at This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at Copyright American Diabetes Association, Inc., 2010

2 Objective: Regulatory T-cells (Tregs) recognizing islet-autoantigens are proposed as a key mechanism in the maintenance of self-tolerance and protection from type 1 diabetes. To date, however, detailed information on such cells in man, and insight into their mechanisms of action, has been lacking. We previously reported that a subset of CD4 T-cells secreting high levels of the immunosuppressive cytokine IL-10 is significantly associated with late onset of T1D and is constitutively present in a majority of non-diabetic individuals. Herein we test the hypothesis that these T-cells represent a naturally generated population of Tregs capable of suppressing proinflammatory T-cell responses. Research Design and Methods: We isolated and cloned islet-specific IL-10 secreting CD4 + T- cells from non-diabetic individuals after brief ex-vivo exposure to islet autoantigens using cytokine capture technology and examined their phenotype and regulatory potential. Results: Islet-specific IL-10+ CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen presenting cell (APC). The regulatory T-cells secrete perforin and granzymes and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells. Conclusions: This hitherto un-described population of islet autoantigen-specific Tregs, displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen beta cellspecific tolerance. 2

3 T here is now overwhelming evidence from both mice and men that regulatory T-cells (Tregs) play a key role in the process of controlling the expansion of pathogenic autoreactive T-cells and that defects in this key cell population can lead to the development of autoimmune disease including T1D (1-4). A view has therefore emerged that in the majority of individuals for the majority of life, destructive autoimmunity is held in check by populations of Tregs, recognizing autoantigens and responding through a variety of regulatory pathways (5-8). Given the apparent importance of this cell type in promoting self-tolerance, there has been much interest in investigating whether strategies to increase the number or functional potency of Tregs are capable of influencing the progression of autoimmune diseases such as T1D. Indeed, numerous studies in animal models have demonstrated that such strategies are not only able to prevent the onset of T1D but are also able to reverse established diabetes, making them a logical target for intervention strategies in humans (9-13). Critically, these studies highlighted the importance of antigen specificity: islet-specific Tregs have far greater potency than those derived from polyclonal populations. This presents a substantial challenge to translating advances in our understanding of Treg biology into immunotherapy for human T1D, since the literature on the natural repertoire of human islet-antigen specific Tregs that may contribute to the tolerant state in vivo is scant. In recent years, we have developed approaches that allow the functional interrogation of human autoreactive T-cells directly ex vivo for their ability to respond to islet autoantigens (14; 15). In a previous study, we reported the existence of a population of islet autoreactive CD4 T-cells that produce the signature immunosuppressive cytokine IL-10 after brief co-culture with naturally processed and presented epitopes of proinsulin and IA-2 (16). Of note, such cells were frequently found in non-diabetic individuals, and when present in patients, they primarily segregated with a significantly older age of onset. We reasoned that such cells could have regulatory properties, since they were associated with both the absence and delayed onset of disease. The present study tests this hypothesis. We isolated and cloned CD4+ T- cells secreting IL-10 after brief ex vivo exposure to islet autoantigens. As subjects, we elected to use islet-autoantibody negative non-diabetic individuals, reasoning that in such individuals immune regulation would be most operative. We find that the islet specific IL-10 secreting CD4+ T-cells are potent suppressors of pro-inflammatory effector Th1 cells, of the type believed to mediate islet β- cell destruction. Surprisingly, despite the cells IL-10 production, we find that their primary modus operandi in vitro is the specific destruction of antigen presenting cells presenting peptides from islet autoantigens. Our study represents an unequivocal demonstration of autoantigen-specific Tregs in human T1D and provides a novel platform for the development of strategies to promote their development. METHODS Subject: Fresh heparinised blood samples were obtained from 11 healthy nondiabetic control subjects with no family history of T1D. Ethical approval for this study was granted by the local Ethics Committee and informed consent obtained. Antigens and synthetic peptides and cytokine ELIspot: Previously identified peptides, representing naturally processed and presented epitopes of IA-2 and proinsulin (16; 17) and a set of overlapping peptides 3

4 encompassing the sequence of human insulin were synthesized by Keck Biotechnology Resource Laboratory, Yale University, USA. Tetanus toxoid was obtained from Pasteur Merieux (Maidenhead, UK), haemagglutinin from Solvay Pharmaceuticals, Inc (Marietta, GA, USA) and human insulin from Sigma Chemical Corporation (Poole, UK). ELISpot assays for the detection of IL-10 producing T- cells were performed and analysed as previously described (16) Isolation and cloning of IL-10 secreting CD4+ T-cells: IL-10 secreting CD4+ T-cells were isolated using the Miltenyi IL-10 secretion, detection and enrichment kit following the manufacturer s protocol (Miltenyi Biotech, Surry, UK). Cells were labeled with monoclonal anti-cd3, - CD4, -CD14 antibodies and 7- aminoactinomycin D (7-AAD, Merck Chemical Ltd., Nottingham, UK), analyzed on a MoFlo flow cytometer (Cytomation Bioinstruments, Freiburg, Germany) and single, viable, IL-10 secreting T-cells deposited into the wells of a 96-well tissue culture plate. Single cells were expanded by the addition of mixed-donor irradiated PBMCs (10 5 /well), PHA-L (4µg/ml; Biostat Diagnostic Systems, Stockport, UK) and human recombinant IL-15 (10ng/ml; Peprotech, London, UK) in 200µl/well complete media (X-Vivo15 +5% pooled human AB serum, BioWhittaker) and tested for antigen-specificity. Antigen-specific clones were restimulated every days and human recombinant IL-15 (10 ng/ml; Peprotech) was added at day 3 and as required during expansion. T-cell clones were subjected to a maximum of 3 cycles of stimulation. T-cell clones were used for the experiments described below when fully rested, a minimum of 21 days post stimulation. Characterization of IL-10 secreting T-cell clones: IL-10 and TNF-α secretion by T-cell clones were measured by ELISA (ImmunoTools, Friesoythe, Germany); all other cytokines were measured by multiplex bead array (Millipore Ltd, Watford, UK) on the Luminex 100 LabMAP System (Luminex Corporation, Oosterhout, NL). Analysis of cytokine production by intracellular flow cytometry, was performed as previously described (18). T-cell clones was studied by flow cytometry, using anti-cd4, -CD3, CD69, - HLA-DR, -CD39, -GITR, -CD62L -α4 integrin, and -β7 integrin antibodies (BD Biosciences, Oxford, UK); anti-icos antibody (Biolegend, San Diego, USA) and anti-cd25 (AbD Serotec, Oxford, UK). FoxP3 staining was performed using the e- Bioscience anti-human Foxp3 Staining Set (Insight Biotechnology Ltd, Wembley, UK) and granzyme and perforin expression were detected using BD Biosciences staining sets, following the manufacturer s instructions. Flow cytometry was performed on a FACSCalibur or FACSAria (BD Biosciences Oxford, UK) and data analyzed using FlowJo software (TreeStar Ltd, Ashland, OR, USA). Suppression assays: Suppression assays were performed through co-culture of putative regulatory T-cell clones with effector populations and APCs that had been differentially labeled with intracellular fluorescent dyes carboxyfluorescein diacetate, succinimidyl ester (CFSE) and CellTrace TM Far Red DDAO-SE (DDAO) (Invitrogen, Paisley, UK). Autologous suppression assays were established in 96-well plates with 1x10 5 CFSE labeled PBMCs/well +/-3x10 4 DDAO labeled Treg clone. To facilitate further analysis of the mechanisms of suppression, a panel of CD4 + Th1 clones specific for recall antigens was generated as detaied in Supplementary Table 1. Clonal suppression assays were established in 96-well plates with 3x10 4 CFSE labeled clone cells, 1x10 5 DDAO labeled irradiated (3000 rad) PBMCs as a source of antigen presenting cells +/- 3x10 4 DDAO labelled Treg clone. To study the role 4

5 of cytokines in suppression, anti-il-10rα (clone ), anti-tgf-β 1,2,3 (clone 1) (both R&D Systems, Abingdon, UK) and mouse IgG1 isotype control (clone MOPC31C; Sigma Chemical Corporation, Poole, UK) antibodies were added to the cultures to a final concentration of 10µg/ml. Transwell assays were established in 24-well plates (Costar; Corning, NY, USA) using 3x10 5 CFSE labeled responder clone, 1x10 5 DDAO labeled irradiated PBMCs and 3x10 5 DDAO labeled Treg clone per well as indicated. Percent suppression was calculated as previously described (19). Cytotoxicity assay: Autologous PBMC were labeled with either 0.1 or 1µM CFSE and incubated in complete media at 1x10 6 /ml for 3 hours at 37 C with 25µg/ml peptide (0.1µM labeled cells) or peptide diluent (DMSO; 1µM labeled cells). Unbound peptide was removed by extensive washing, target populations combined at a ratio of 1:1 and 10 5 PBMC incubated in the presence or absence of 10 5 resting Treg clones. Following incubation for 16 hr at 37 C, cells were stained with anti-cd4, anti- CD14 and anti-cd19 fluorochrome-labeled antibodies (BD Biosciences Oxford, UK) and 7-AAD and analysed by flow cytometry and percent specific killing calculated as: 100-((% peptide-pulsed cells/% unpulsed cells)*100). In experiments using EBV B-cell lines, target populations were pulsed with peptide as described above and killing assessed by calculating the percentage of 7- AAD+ve cells. To assess the role of the granzyme/perforin and Fas/Fas ligand (FasL) pathways in killing, cells were treated with ethylene glycol tetraacetic acid (EGTA, 4mM, Merck), Concanamycin A (100nM, Sigma Chemical Corporation), anti-cd95l antibody (1µg/ml ZB4, Immunotech, France). Single cell multiplex RT-PCR: Multiplex RT-PCR was performed on single IL-10 secreting CD4 T-cells isolated from fresh PBMC preparations after co-culture with islet autoantigen as described above for cloning, based on the technique described by Kelso and colleagues (20), using the primer pairs detailed in Supplementary Table 2. The frequency of expression of cytotoxic molecules in different groups was compared by ( 2 analysis. RESULTS Detection and cloning of islet peptide-specific IL-10 secreting T-cells from non-diabetic individuals. We first examined IL-10 responses to a panel of naturally processed IA-2 and proinsulin peptides and a set of overlapping peptides encompassing the insulin molecule in a group of healthy non-diabetic individuals (n=11). Utilizing a highly sensitive and specific cytokine ELISpot, we found that the majority of individuals (8/11, 72%) mounted a significant IL-10 response (SI 3) to at least one peptide (data not shown), consistent with our previous work (16). Individuals with positive responses by ELISpot were recalled a minimum of one month later, and IL-10- secreting CD4 T-cells detected, isolated by sensitive cytokine secretion assay (CSA) and immediately single cell sorted by flow cytometry. The CSA confirmed the reproducible detection of IL-10 secreting, islet peptide specific CD3 + CD4 + T-cells in non-diabetic individuals (Figure 1). Cloning was initiated after brief (48 hour) culture with antigen and was carried out in the absence of any IL-10 biasing culture conditions. T-cell clones specific for insulin B11-30 (clone MHB10.3) and IA (clone RAR5.3) were isolated from non-diabetic individuals, MH (age 27 years; HLA-DRB1*0301- DRB1*0404) and RA (age 27 years; HLA- DRB1*0101-DRB1*0407) respectively. Both clones produced large amounts of IL-10 (>1 ng/ml), in response to sub-microgram doses of the peptide used in their isolation (Figure 2A). 5

6 Phenotypic analysis of IL-10 secreting islet-specific T-cells: Analysis of cultures stimulated with suboptimal doses of peptide demonstrated that both clones have a similar cytokine secretion profile, producing large amounts of IL-10, IL-13, IFN-γ and IL- 5 but little or no IL-2, IL-4, TNF-α or IL-1β in (Figure 2B). A similar cytokine secretion profile, was seen when MHB10.3 was incubated with whole recombinant insulin (Figure 2C). Analysis of cytokine production at a single cell level shows that when activated and producing cytokines, the same cells produce IL-10, IFN- and IL-13 (Figure 2D). The HLA-class II restriction of the clones was investigated using monoclonal antibodies that block HLA-DR or HLA-DQ and partially HLA-matched irradiated PBMCs. These experiments demonstrated that clone MHB10.3 was restricted by HLA- DR3 (B1*0301) and clone RAR5.3 by HLA- DR4 (DRB1*0407) (data not shown). Expression of activation and regulatory T-cell markers on islet specific T- cells was assessed by flow cytometry. A small number of resting IL-10 secreting islet specific clone cells (21 days post peptide stimulation) expressed a low level of CD69 (14.4% and 7.5% for RAR5.3 and MHB10.3, respectively) whilst much higher proportions expressed HLA-DR (94.6% and 60.4%), CD25 (99.8% and 92.8%), FoxP3 (97.5% and 61.9%), CD39 (100% and 99.8%), inducible co-stimulator (ICOS; 92% and 97.2%), lower levels of glucorticoid-induced TNF receptor (GITR; 42.2% and 23.4%) and CD62L (10.5% and 18.7%) and variable amounts of CD127 (32.7% and 64.1%) (Supplementary Figure 1 which can be found in an online appendix at In addition both MHB10.3 and RAR5.3 expressed both α4 and β7 integrins (Supplementary Figure 2), which were absent on the TT-specific RATT6 and HA-specific RAHA5 Th1-type T-cell clones. Regulatory potential of IL-10 secreting islet-specific T-cells. To investigate the regulatory potential of IL-10 secreting islet-specific T-cells, a dual fluorescence based suppression assay was used. Regulation was first assessed in an autologous assay system, measuring the response of PBMCs to recall antigen HA. Individual MH exhibited a robust proliferative response to HA (9.6% of CD3 + PBMCs; Figure 3B). Addition of either T-cell clone MHB10.3 or its epitope, Ins B11-30 had little effect on proliferation (Figures 3C-D), whereas addition of MHB10.3 and its epitope InsB11-30 together resulted in complete suppression of the HA-specific proliferative response (Figure 3E). These data demonstrate that IL- 10 secreting islet-specific T-cells are potent regulators, and that regulation is dependent upon activation of Tregs by APC presentation of cognate peptide. To pursue further analysis of the mechanisms of suppression, we used a panel of CD4 + Th1 clones specific for recall antigens TT and HA (RATT6 and RAHA5, respectively). Both clones proliferate rapidly in response to cognate antigen with 68% and 52% of cells divided after 3 days respectively (Figure 4B&E). However, when cultured with MHB10.3 (Figure 4C) or RAR5.3 (Figure 4F) in the presence of the cognate islet-peptide, proliferation was dramatically reduced (33% and 16%) resulting in suppression rates of 51% and 69% respectively. Blocking IL-10 and TGF-β either individually or in combination had no effect on the ability of MHB10.3 or RAR5.3 to suppress proliferation of RATT6 (74-80% suppression, Figure 5A-H) or RAHA5 (51-59% suppression, Figure 5I). This absence of IL-10 dependency of regulation was also present in studies in which Treg number and peptide concentration were titrated to extinction of activity (Supplementary Figure 3). Furthermore, blockade of IL-10 during the activation and expansion of IL-10 secreting islet-specific T-cell clones also did not affect 6

7 the ability of the cells to regulate (Supplementary Figure 4). However, IL-10 secreted by these Tregs was found to upregulate monocyte expression of the molecule B7-H4 (a B7 family member that negatively regulates T cell immunity through inhibition of T cell proliferation, cytokine production, and cell cycle progression (21)) and that this is inhibited by anti-il-10r antibody (Supplementary Figure 5). IL-10 secreting islet-specific Tregs require cell-cell contact to mediate suppression. We used a transwell system to investigate whether suppression by IL-10 secreting islet-specific -Tregs is mediated by soluble factors or dependent on cell-cell contact. When activated by peptide, MHB10.3 is able to suppress proliferation of RATT6 in co-culture, but this is completely ablated if responder and regulatory T-cells are physically separated (Figure 6 A-D). The role of the APC in suppression was then more closely investigated. First, a standard suppression assay was established using irradiated PBMCs from a donor with both HLA-DRB1*0301 and HLA-DRB1*0407 genotypes, that can therefore present antigens to both regulatory (MHB10.3) and responder (RATT6) T-cell clones (Figures 6E-F). Second, assays were established using irradiated PBMCs from two different donors, (one with the HLA-DRB1*0301 genotype but not HLA-DRB1*0407 and another with the HLA-DRB1*0407 genotype but not HLA- DRB1*0301) in which presentation to regulatory and responder clones is therefore separated on two different APC populations (Figures 6G-H). Whereas suppression occurs when both responder and regulatory T-cell clones are stimulated by the same APC (Figures 6E-F), no suppression is seen when presentation to regulatory and responder clones takes place on two different APC populations (Figures 6G-H). The lack of suppression in these latter cultures was observed at both high and low levels of effector clone stimulation strength (see Supplementary Figure 6). Similar assays were established using IL-10 secreting isletspecific Treg RAR5.3 using two independent responder T-cell clones and sources of APCs with identical results (Figures 6I&J). IL-10 secreting islet-specific Tregs express cytotoxic molecules and kill islet antigen presenting cells. Since IL-10 secreting islet-specific -Treg-mediated suppression requires cell-cell contact and is not mediated by cytokines conventionally associated with regulation, we sought alternative mechanisms to account for these effects. We noted that resting IL-10 secreting islet-specific -Treg clones express granzymes A and B and perforin and that expression of perforin and granzyme B was upregulated upon activation (Supplementary Figure 7). To investigate whether expression of these cytotoxic molecules translated into an ability to kill APCs, a flow-cytometry based cytotoxicity assay was employed. High levels of cytoxicity were seen against APCs loaded with islet autoantigen (56% at an effector:target ratios of 10:1), whereas APCs lacking islet peptide were not killed, even at high effector:target ratios (Figure 7A). In order to investigate the specificity of killing, a dual fluorescence-intensity killing assay was used. In this assay peptide pulsed and unpulsed PBMCs were incubated in the same culture along with IL-10 secreting isletspecific -Tregs. In these experiments only peptide-loaded APCs (i.e. CD19 + B cells and CD14 + monocytes) were killed (specific killing of 80% and 65% respectively) whilst APCs lacking islet peptide and CD4 + T-cells were not killed despite juxtaposition with highly activated Tregs in the cultures (Figure 7B). In order to investigate the mechanism of killing, cells were pre-incubated with inhibitors of the perforin pathway (ConA or EGTA), or Fas/FasL ligand pathway (neutralizing anti-fasl antibody) (Figure 7

8 7C&D). Whereas killing was unaffected in the presence of anti-fasl antibodies, it was completely abrogated in the presence of ConA or EGTA demonstrating that IL-10 specific islet-specific Tregs exhibit perforingranzyme dependent cytotoxicity. Using cell viability dyes we demonstrate that during assays in which Tregs are activated by peptide-loaded APCs, the responder T cells undergoing regulation are not themselves killed (Supplementary Figure 8). The kinetics of suppression in relation to responder T cell proliferation are shown in Supplementary Figure 9 and demonstrate that suppression is apparent and most potent when measured at Day 2 (the earliest timepoint at which proliferation of responder T cells can be detected; Supplementary Figure 9A). Furthermore if Tregs are placed in the APC/peptide culture for 24 hours prior to addition of the T responders suppression is greatly enhanced, presumably due to killing of most APCs before they have the chance to stimulate responder T cells (Supplementary Figure 9B-D). IL-10 secreting islet autoantigenspecific Tregs express cytotoxic molecules directly ex-vivo. In order to establish whether IL-10 secreting islet-specific Tregs express cytotoxic molecules directly ex-vivo, or gain this expression as a consequence of the cloning process in vitro, we performed a multiplex RT-PCR on single IL-10 + CD4 + T- cells freshly isolated from islet peptide stimulated PBMC cultures (Figure 8). These experiments were performed with the original donors used to clone MHB10.3 and RAR5.3 Tregs. All sorted T-cells were examined for CD3 expression as a template quality control and also for IL-10 expression. Only IL-10 + sorted T-cells that were also IL-10 transcript positive were included in the analysis. These studies demonstrated that 67% (6/9) of the IL- 10 transcript positive T-cells expressed at least one cytotoxic molecule, compared with 5.6% (1/18) of the IL-10 transcript negative T-cells (p=0.0006). DISCUSSION The present study builds upon our previous observation that in type 1 diabetes, a distinction can be made between tolerant and disease states in terms of the polarization of islet-specific CD4 T-cell responses. Notably, we previously showed that islet autoreactive T-cells identified by IL-10 secretion are associated with both the non-diabetic state and late onset disease. On this basis, we proposed that IL-10 production by CD4 T- cells in response to islet autoantigens represents a signature of immune regulation. As proof of this concept, in the present study we report the first cloning of such human, interleukin-10 secreting islet-specific T cells (which we term ISIS Tregs) and demonstrate that their marked capacity for linked suppression of pro-inflammatory T-cells is associated with APC killing. We first screened a cohort of healthy individuals to identify those with robust IL-10 polarization of islet autoreactive CD4 T-cell responses. In agreement with our earlier studies, we found that the majority of nondiabetic individuals mounted a significant IL- 10 response to at least one islet autoantigen. We next employed a sensitive, direct, ex vivo cloning strategy to isolate and expand the IL- 10-producing cells, in order to derive sufficient numbers with which to examine our original hypothesis that they have regulatory properties. We demonstrate that not only are IL-10 secreting islet-specific T-cells capable of suppressing responses of resting CD4 memory T-cells present in PBMC preparations, they are also potent suppressors of both the proliferation and cytokine production of highly activated Th1 T-cell clones similar to those believed to play a role in the pathogenesis of T1D. In contrast, in our experience neither freshly isolated nor expanded populations of the prototypic 8

9 polyclonal CD4 + CD127 lo CD25 + ntregs are able to suppress activated Th1 clones to such a degree (J Lawson personal communication). Further examination of their function revealed that ISIS-Tregs have a highly distinctive mode of action. Their ability to suppress effector T-cell function is mediated entirely independently of production of cytokines or any other paracrine soluble factors, requiring instead direct-cell contact. Using ISIS-Treg and effector T-cell clones with differing HLA-restriction elements, we limited antigen presentation to occur on the same APC or on two different APC populations. Suppression was dependent upon presentation of the cognate antigen for both ISIS-Treg and effector T cells on the same APC. In investigating the molecular and cellular interactions required for this effect, we noted that ISIS-Tregs express perforin and granzymes A and B and therefore examined the APC killing potential of these cells. Only APCs bearing cognate antigen were killed by ISIS-Tregs in a perforin dependent manner, and as a result APC-mediated activation of effector clones was effectively negated. Critically, we confirmed expression of perforin and granzymes in freshly isolated ISIS-Tregs directly ex-vivo, excluding the possibility that their expression is an artefact of clonal expansion in vitro. Based on our studies in vitro, we hypothesize that in vivo, ISIS-Tregs achieve regulation by a kill the messenger approach in which APCs displaying high levels of selfantigens are rapidly deleted, thus preventing the priming and/or expansion of potentially pathogenic autoreactive CD4 T helper and CD8 cytotoxic T-cells. We propose a threecell model of linked suppression in which professional APCs will display peptide-hla complexes representing a range of autoantigens including insulin, IA-2 and glutamic acid decarboxylase-65 released by beta cells (perhaps damaged by an environmental insult such as a virus (22)). ISIS Tregs will kill these APCs, whilst sparing bystander APCs not displaying isletantigens. Whilst not providing classical antigen specific regulation (i.e. only regulating T-cells responding to the same antigen) this mechanism provides tissuespecific regulation, including suppression of T-cells recognizing other islet autoantigens. Such a mechanism of suppression has several important attributes, most notably the highly specific nature and location of regulation as a means of maintaining tissue-specific tolerance. In sum, our data appear to support a model in which the most potent regulatory function of these Tregs in vitro requires cellcell contact and is independent of IL-10. However, as also pointed out by others in this field (23; 24) and supported by our finding in relation to B7-H4, we cannot exclude the possibility that there are additional pathways via IL-10 secretion through which ISIS-Tregs are able to regulate in vivo and these remain to be elucidated. Expression of cytotoxic molecules has been reported in other natural and adaptive human polyclonal Treg populations (25-28). However, comparison with the highly specific killing mediated by ISIS-Tregs is difficult as these other Tregs are of unknown antigenspecificity and require polyclonal stimuli to mediate their suppressor function. The importance of the cytotoxic function of Tregs has recently been demonstrated in a model of transplantation in which Tregs from granzyme B deficient mice were unable to mediate transplant-tolerance (29). Furthermore, Benoist and colleagues have also highlighted the importance of a population of CD25+ Tregs characterised by the expression of IL- 10 and granzyme B in preventing disease in a mouse model of type 1 diabetes (30), supporting the suggestion that cytotoxic ISIS- Tregs may play an important role in maintaining islet-specific tolerance in vivo. At present it is not possible to be certain where ISIS-Tregs stand in the current, 9

10 complex taxonomy of regulatory T-cells. ISIS-Tregs share some features in common with other Treg populations (e.g. constitutive expression of CD25, FoxP3, CD39 and ICOS, requirement for cell-cell contact to mediate suppression, expression of cytotoxic effector molecules) but not others (eg expression of GITR and absence of CD127) and the relevance of these findings to their function and ontogeny will require further investigation. We speculate that ISIS-Tregs are generated in the periphery as a consequence of low-level exposure to autoantigens occurring early during development or chronically, in the absence of inflammatory signals, as has been suggested for the generation of peripheral tolerance (31). Intriguingly, ISIS-Tregs do bear striking similarities (IL-10 secretion and dependence on cell-cell contact) to Tregs generated from naïve cord blood cells by repeated stimulation with immature allogeneic dendritic cells (32), suggesting the possibility that ISIS-Treg may be generated early in development. In summary, we have isolated and characterized a novel population of human islet autoantigen-specific regulatory T-cells associated with late development of type 1 diabetes. These ISIS-Tregs are potent suppressors of Th1 effector cells and mediate suppression via direct contact with autoantigen presenting APCs, providing exquisite specificity of suppression. This hitherto un-described population of antigen specific Tregs, which appears to be constitutively present in the majority of nondiabetic individuals, may provide a suitable target to strengthen tolerance to islets by clinical immunointervention. ACKNOWLEDGEMENTS This work was supported by Diabetes UK and the Sir Jules Thorn Charitable Trust. TT is a Diabetes UK RD Lawrence Research Fellow and JL is the recipient of a Diabetes UK PhD studentship. The authors TT, AS and MP acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London. We are grateful to subjects for blood donation especially MH, RA and TFBH; to Adrian Hayday and Maria Grazia Roncarolo for helpful discussions; and to Wayne Turnbull for performing flow cytometry and cell sorting. 10

11 REFERENCES 1. Sakaguchi S: Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22: , Sakaguchi S, Fukuma K, Kuribayashi K, Masuda T: Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J Exp Med 161:72-87, Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155: , Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, Levy-Lahad E, Mazzella M, Goulet O, Perroni L, Bricarelli FD, Byrne G, McEuen M, Proll S, Appleby M, Brunkow ME: X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 27:18-20, Baecher-Allan C, Hafler DA: Human regulatory T cells and their role in autoimmune disease. Immunol Rev 212: , Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, Fehervari Z, Shimizu J, Takahashi T, Nomura T: Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 212:8-27, Roncarolo MG, Levings MK: The role of different subsets of T regulatory cells in controlling autoimmunity. Curr Opin Immunol 12: , Bach JF: Regulatory T cells under scrutiny. Nat Rev Immunol 3: , Masteller EL, Warner MR, Tang Q, Tarbell KV, McDevitt H, Bluestone JA: Expansion of functional endogenous antigen-specific CD4+CD25+ regulatory T cells from nonobese diabetic mice. J Immunol 175: , Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, Bluestone JA: In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 199: , Tarbell KV, Petit L, Zuo X, Toy P, Luo X, Mqadmi A, Yang H, Suthanthiran M, Mojsov S, Steinman RM: Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. J Exp Med 204: , Tarbell KV, Yamazaki S, Olson K, Toy P, Steinman RM: CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J Exp Med 199: , Staeva-Vieira T, Peakman M, von Herrath M: Translational mini-review series on type 1 diabetes: Immune-based therapeutic approaches for type 1 diabetes. Clin Exp Immunol 148:17-31, Peakman M, Stevens EJ, Lohmann T, Narendran P, Dromey J, Alexander A, Tomlinson AJ, Trucco M, Gorga JC, Chicz RM: Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4. J Clin Invest 104: , Schloot NC, Meierhoff G, Karlsson Faresjo M, Ott P, Putnam A, Lehmann P, Gottlieb P, Roep BO, Peakman M, Tree T: Comparison of cytokine ELISpot assay formats for the detection of islet antigen autoreactive T cells. Report of the third immunology of diabetes society T-cell workshop. J Autoimmun 21: ,

12 16. Arif S, Tree TI, Astill TP, Tremble JM, Bishop AJ, Dayan CM, Roep BO, Peakman M: Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health. J Clin Invest 113: , Di Lorenzo TP, Peakman M, Roep BO: Translational Mini-Review Series on Type 1 Diabetes: Systematic analysis of T cell epitopes in autoimmune diabetes. Clin Exp Immunol 148:1-16, Skowera A, Cleare A, Blair D, Bevis L, Wessely SC, Peakman M: High levels of type 2 cytokine-producing cells in chronic fatigue syndrome. Clin Exp Immunol 135: , Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TI: Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes 54:92-99, Kelso A, Groves P, Ramm L, Doyle AG: Single-cell analysis by RT-PCR reveals differential expression of multiple type 1 and 2 cytokine genes among cells within polarized CD4+ T cell populations. Int Immunol 11: , Sica GL, Choi IH, Zhu G, Tamada K, Wang SD, Tamura H, Chapoval AI, Flies DB, Bajorath J, Chen L: B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 18: , Dotta F, Censini S, van Halteren AG, Marselli L, Masini M, Dionisi S, Mosca F, Boggi U, Muda AO, Prato SD, Elliott JF, Covacci A, Rappuoli R, Roep BO, Marchetti P: Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients. Proc Natl Acad Sci U S A 104: , Wraith DC: Role of interleukin-10 in the induction and function of natural and antigeninduced regulatory T cells. J Autoimmun 20: , O'Garra A, Vieira PL, Vieira P, Goldfeld AE: IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage. J Clin Invest 114: , Gondek DC, Lu LF, Quezada SA, Sakaguchi S, Noelle RJ: Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforinindependent mechanism. J Immunol 174: , Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ: Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity 21: , Grossman WJ, Verbsky JW, Tollefsen BL, Kemper C, Atkinson JP, Ley TJ: Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood 104: , Kawamura K, Kadowaki N, Kitawaki T, Uchiyama T: Virus-stimulated plasmacytoid dendritic cells induce CD4+ cytotoxic regulatory T cells. Blood 107: , Gondek DC, Devries V, Nowak EC, Lu LF, Bennett KA, Scott ZA, Noelle RJ: Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells. J Immunol 181: , Herman AE, Freeman GJ, Mathis D, Benoist C: CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 199: , Carbone FR, Belz GT, Heath WR: Transfer of antigen between migrating and lymph noderesident DCs in peripheral T-cell tolerance and immunity. Trends Immunol 25: , Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH: Induction of interleukin 10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 192: ,

13 Figure Legends. Figure 1. Representative IL-10 responses to islet antigen peptides by PBMCs from non-diabetic individuals stimulated with (A) insulin B11-30 peptide compared with (B) peptide diluent alone in non-diabetic individual MH; (C) IA peptide compared with (D) peptide diluent alone in non-diabetic individual RA. (E-F) Isolation of IL-10 secreting cells by cytokine secretion assay. Plots represent flow cytometry analysis of magnetically enriched IL-10 secreting cells from MH stimulated with insulin B11-30 peptide (E) compared with background response to (F) diluent alone. Figure 2. Cytokine production profiles of islet specific T-cell clones. (A) MHB10.3 (open symbols) and RAR5.3 (closed symbols) were stimulated with insulin B11-30 and IA , respectively in the presence of autologous irradiated PBMCs and IL-10 production measured by ELISA after 3 days. (B) Cytokine production by MHB10.3 (open bars) and RAR5.3 (closed bars) clones cultured as above with suboptimal doses of peptide (1µg/ml) and by (C) MHB10.3 stimulated with whole recombinant insulin (100µg/ml) in the presence of autologous irradiated dendritic cells. Cytokines were measured in 3-day supernatants by multiplex bead technology. (D) Cytokine production by MHB10.3 was assessed by flow cytometry for intracellular synthesis of IL-10, IL-13 and interferon-γ after stimulation with plate bound anti-cd3 and soluble anti- CD28 antibodies for 2 days. Figure 3. Regulation of autologous T-cell responses by MHB10.3. A-E PBMCs from MH were labeled with CFSE and stimulated with combinations of recombinant influenza haemagglutinin (HA; 45ng/ml) and insulin B (10µg/ml) and in the presence or absence of DDAO labeled MHB10.3 IL-10 secreting islet specific T cell clone as indicated. Proliferation of CD3+ T-cells was assessed after 6 days by flow cytometry. Numbers represent the % CD3+ PBMC in the gated area. Data are representative of more than three independent experiments. When activated by cognate antigen, clone MHB10.3 completely abrogates the proliferative response of recall memory T cells specific for HA. Figure 4. Regulation of clonal T-cell responses by MHB10.3 and RAR5.3. A-C The tetanus toxoid specific Th1 clone RATT6 was labelled with CFSE and stimulated with combinations of tetanus toxoid (100ng/ml) and insulin B (10µg/ml) in the presence or absence of DDAO labeled MHB10.3 as indicated. D-F The haemagglutinin specific Th1 clone RAHA5 was labeled with CFSE and stimulated with combinations of recombinant haemagglutinin (45ng/ml) and IA (25µg/ml) in the presence or absence of DDAO labeled RAR5.3 as indicated. HLAmatched DDAO labeled irradiated PBMCs were used as a source of antigen presenting cells. Proliferation of Th1 clones (DDAO-ve cells) was assessed after 3 days by flow cytometry and the gated regions represent the percent of live clone cells that have undergone division. Proliferation of the Th1 clones RATT6 and RAHA5 is suppressed by activated MHB10.3 and RAR5.3 Treg clones. Data are representative of a minimum of three independent experiments. Figure 5. Regulation of clonal T-cell responses by IL-10 secreting islet-specific Tregs are not dependent on IL-10 or TGF-β. A-H CFSE labeled RATT6 and HLA-matched DDAO labeled irradiated PBMCs were stimulated with tetanus toxoid (100ng/ml) and insulin B (10µg/ml) in the absence (A-D) or in the presence (E-H) of DDAO labeled MHB10.3 and monoclonal 13

14 antibodies as indicated. Proliferation of RATT6 (DDAO-ve cells) was assessed after 3 days by flow cytometry. Numbers indicate the percentage suppression in the presence of MHB10.3. I A similar experiment in which CFSE labeled RAHA5, DDAO labeled RAR5.3 and HLA-matched DDAO labeled irradiated PBMCs were stimulated with either haemagglutinin (45ng/ml) alone (black bars) or haemagglutinin and IA (25µg/ml) (white bars) in the presence of monoclonal antibodies as indicated. Proliferation of RAHA5 (DDAO-ve cells) was assessed after 3 days by flow cytometry. Numbers indicate the percentage suppression in the presence of IA and error bars indicate standard error from the mean for triplicate cultures. Data are representative of at least three independent experiments. Figure 6. Regulation of clonal T-cell responses by IL-10 secreting islet-specific Tregs is dependent on cell-cell contact and operates by a linked suppression mechanism. A-D. CFSE labeled RATT6 and DDAO labeled MHB10.3 were stimulated with tetanus toxoid (100ng/ml) or tetanus toxoid and insulin B (10µg/ml) in the upper or lower chamber of a transwell-plate as indicated. HLA-matched DDAO labeled irradiated PBMCs as a source of antigen presenting cells were present in both upper and lower chambers. Proliferation of RATT6 (DDAO-ve cells) was assessed after 4 days by flow cytometry. E-H. CFSE labeled RATT6 (HLA-DRB1*0407 restricted) Th1 clone cells were stimulated with tetanus toxoid (100ng/ml) and insulin B (10µg/ml) in the presence or absence of DDAO-labeled MHB10.3 (HLA-DRB1*0301 restricted) Treg clone as indicated. Partially or fully HLA matched DDAO labeled irradiated PBMCs were used as a source of antigen presenting cells as indicated. Proliferation of RATT6 (DDAO-ve cells) was assessed after 4 days by flow cytometry. I-J. The HLA-DRB1*0101 restricted Th1 clone RAHA5 (I) and the HLA-DRB1*0401 restricted Th1 clone TTTT6 (J) were labeled with CFSE and stimulated with haemagglutinin (45ng/ml) or tetanus toxoid (100ng/ml) respectively in the presence or absence of DDAO-labeled RAR5.3 (HLA-DRB1*0407 restricted) Treg clone and IA (25µg/ml) as indicated. Partially or fully HLA matched DDAO labeled irradiated PBMCs were used as a source of antigen presenting cells as indicated. Proliferation of Th1 clones (DDAO-ve cells) was assessed after 4 days by flow cytometry. Numbers indicate % suppression and error bars indicate standard error from the mean for triplicate cultures. Figure 7. Islet peptide pulsed antigen presenting cells are specifically killed by islet specific Tregs. A. The HLA-DRB1*0401 EBV-B cell line, Preiss was labeled with CFSE and either pulsed with IA (filled symbols) or peptide diluent alone (open symbols) and then incubated with varying E:T ratios with resting RAR5.3 T-cells. Death of CFSE + antigen presenting cells was assessed by uptake of 7-AAD. Similar results were obtained with MHB10.3. B. Autologous PBMC from individual RA were labeled with either 0.1µM CFSE and pulsed with IA or 1µM CFSE and pulsed with DMSO diluent alone. After 3 hours excess peptide was removed by rigorous washing and the peptide pulsed and unpulsed PBMCs were combined at a 1:1 ratio and incubated either alone or with resting RAR5.3 T-cells as indicated. Following incubation for 16 hr at 37 C, cultures were harvested and death in different cell populations assessed by multi-color flow cytometry. Numbers indicate % specific death of peptide pulsed cells in the gated population. C&D. Autologous PBMC from individuals RA (C) and MH (D) were labeled with CFSE and pulsed with IA and Ins B11-30 respectively or DMSO as described for panel B. Peptide pulsed and unpulsed PBMCs were combined at a 1:1 ratio and incubated either alone (black bars) or with resting IL-10 secreting islet-specific Tregs (white bars) in the presence of inhibitors of cytotoxicity as indicated. Following incubation for 14

15 16 hr at 37 C cultures were harvested and death in different cell populations assessed by multicolor flow cytometry. Plots show % of viable CD19+ lymphocytes that were peptide pulsed. Numbers indicate % specific death of peptide pulsed cells in the gated population and error bars indicate standard error from the mean for triplicate cultures. Results indicate inhibition of APC killing by Tregs in the presence of inhibitors of cytotoxic granule release. Data are representative of at least three independent experiments. Figure 8 IL-10 producing islet specific CD4 + T-cells express cytotoxic molecules directly ex vivo. PBMCs from donors MH and RA were stimulated with islet peptides, and after 48 hours single CD4 + IL or CD4 + IL-10 - T-cells isolated by flow cytometry were deposited into 96-well plates containing 8µl lysis buffer. Reverse transcription and first round PCR (30 cycles) were performed using gene specific primers and the One Step RT-PCR kit following the manufacturer s instructions (Qiagen Ltd., Crawley, UK). Following multiplex RT-PCR, 1µl of a 1:100 dilution of the reaction was used in individual gene, second round, nested PCR reactions (30 cycles) using internal primers and HotStar Taq Polymerase kit (Qiagen). Products were analysed by agarose gel electrophoresis. Data were only considered for analysis from cells that yielded a CD3ε product. Each lane represents products amplified from a single cell. As a positive control, pools of 5 cells of the clones RAR5.3 and MHB10.3 were used following peptide stimulation. 15

16 Figure 1 16

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18 Figure 5 18

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21 FIGURE 8 CD3 IL-10 Granzyme A Granzyme B Perforin 21

There is now overwhelming evidence from both

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